Methods for screening cellular proliferation using isotope labels

ABSTRACT

The present invention relates to methods for measuring the proliferation and destruction rates of cells by measuring deoxyribonucleic acid (DNA) synthesis. In particular, the methods utilize non-radioactive stable isotope labels to endogenously label DNA synthesized through the de novo nucleotide synthesis pathway in a cell. The amount of label incorporated in the DNA is measured as an indication of cellular proliferation. Such methods do not involve radioactivity or potentially toxic metabolites, and are suitable for use both in vitro and in vivo. Therefore, the invention is useful for measuring cellular proliferation in humans for the diagnosis of a variety of disease conditions in which cellular proliferation is involved.

The present invention relates to methods for measuring the proliferationand destruction rates of cells by measuring deoxyribonucleic acid (DNA)synthesis. In particular, the methods utilize non-radioactive stableisotope labels to endogenously label DNA synthesized through the de novonucleotide synthesis pathway in a cell. The amount of label incorporatedin the DNA is measured as an indication of cellular proliferation. Suchmethods do not involve radioactivity or potentially toxic metabolites,and are suitable for use both in vitro and in vivo. Therefore, theinvention is useful for measuring cellular proliferation in humans forthe diagnosis of a variety of disease conditions in which cellularproliferation is involved.

BACKGROUND OF THE INVENTION

Control of cell proliferation is important in all multicellularorganisms. A number of pathologic processes, including cancer andacquired immunodeficiency syndrome (AIDS) (Ho et al., 1995, Nature373:123-126; Wei et al., 1995, Nature 373:117-120; Adami et al., 1995,Mutat. Res. 333:29-35), are characterized by failure of the normalregulation of cell turnover. Measurement of the in vivo turnover ofcells would therefore have wide applications, if a suitable method wereavailable. Prior to the present invention, direct and indirecttechniques for measuring cell proliferation or destruction existed, butboth types were flawed.

Direct measurement of cell proliferation generally involves theincorporation of a labeled nucleoside into genomic DNA. Examples includethe tritiated thymidine (³H-dT) and bromodeoxyuridine (BrdU) methods(Waldman et al., 1991, Modern Pathol. 4:718-722; Gratzner, 1982, Science218:474-475). These techniques are of limited applicability in humans,however, because of radiation induced DNA damage with the former (Asheret al., 1995, Leukemia and Lymphoma 19:107-119) and toxicities ofnucleoside analogues (Rocha et al., 1990, Eur. J. Immunol. 20:697-708)with the latter.

Indirect methods have also been used in specific cases. Recent interestin CD4⁺ T lymphocyte turnover in AIDS, for example, has been stimulatedby indirect estimates of T cell proliferation based on their rate ofaccumulation in the circulation following initiation of effectiveanti-retroviral therapy (Ho et al., 1995, Nature 373:123-126; Wei etal., 1995, Nature 373:117-120). Unfortunately, such indirect techniques,which rely on changes in pool size, are not definitive. The increase inthe blood T cell pool size may reflect redistribution from other poolsto blood rather than true proliferation (Sprent and Tough, 1995, Nature375:194; Mosier, 1995, Nature 375:193-194). In the absence of directmeasurements of cell proliferation, it is not possible to distinguishbetween these and other (Wolthers et al., 1996, Science 274:1543-1547)alternatives.

Measurement of cell proliferation is of great diagnostic value indiseases such as cancer. The objective of anti-cancer therapies is toreduce tumor cell growth, which can be determined by whether tumor DNAis being synthesized or being broken down. Currently, the efficacy oftherapy, whether chemotherapy, immunologic therapy or radiation therapy,is evaluated by indirect and imprecise methods such as apparent size byx-ray of the tumor. Efficacy of therapy and rational selection ofcombinations of therapies could be most directly determined on the basisof an individual tumor's biosynthetic and catabolic responsiveness tovarious interventions. The model used for bacterial infections inclinical medicine—culture the organism and determine its sensitivitiesto antibiotics, then select an antibiotic to which it is sensitive—couldthen be used for cancer therapy as well. However, current managementpractices proceed without the ability to determine directly how well thetherapeutic agents are working.

A long-standing vision of oncologists is to be able to selectchemotherapeutic agents the way antibiotics are chosen—on the basis ofmeasured sensitivity to each drug by the tumor of the patient inquestion. The ability to measure cancer cell replication would placechemotherapy selection and research on an equal basis as antibioticselection, with great potential for improved outcomes.

Accordingly, there remains a need for a generally applicable method formeasuring cell proliferation that is without hazard and can be appliedin the clinical arena.

SUMMARY OF THE INVENTION

The present invention relates to methods for measuring cellularproliferation and destruction rates by measuring DNA synthesis. Inparticular, it relates to the use of a non-radioactive stable isotopelabel to endogenously label DNA synthesized by the de novo nucleotidesynthesis pathway in a cell. The label incorporated into the DNA duringDNA synthesis is readily detectable by methods well known in the art.The amount of the incorporated label can be measured and calculated asan indication of cellular proliferation and destruction rates.

The invention is based, in part, on the Applicants' discovery that DNAsynthesis can be measured by labeling the deoxyribose ring with a stableisotope label through the de novo nucleotide synthesis pathway. Cellularproliferation was measured in vitro, in an animal model and in humans.In vitro, the proliferation of two cell lines in log phase growth wasmeasured by the methods of the invention and was shown to be in closequantitative agreement with the increased number of cells by direct cellcounting, which is considered the least ambiguous measure of cellproliferation. In animals, the methods of the invention were also shownto be consistent with values estimated previously by independenttechniques. For example, thymus and intestinal epithelium were shown tobe rapid turnover tissues, while turnover of liver cells was muchslower. In humans, the observed pattern of a lag phase followed by rapidappearance of a cohort of labeled granulocytes is also consistent withprevious observation.

The methods differ from conventional labeling techniques in 3 majorrespects. First, conventional isotopic methods label DNA through thenucleoside salvage pathway, whereas the methods of the invention labeldeoxyribonucleotides in DNA by the de novo nucleotide synthesis pathway(FIG. 1). Labeling via this pathway is advantageous because in mostcells that enter the S-phase of the cell cycle, the key enzymescontrolling de novo synthesis of deoxyribonucleotide-triphosphates(dNTP's), in particular ribonucleotide reductase (RR), are upregulated,whereas the enzymes of the nucleoside salvage pathway are suppressed(Reichard, 1978, Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem.57:349-374; Cohen et al., 1983, J. Biol. Chem. 258:12334-12340).

Second, the label can be detected in the methods of the invention inpurine deoxyribonucleosides instead of pyrimidines (e.g. from ³H-dT orBrdU). This is advantageous because the de novo synthesis pathway tendsto be more active for purine than pyrimidine dNTP's (Reichard, 1978,Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem. 57:349-374; Cohenet al., 1983, J. Biol. Chem. 258:12334-12340). In fact, regulatorydeoxyribonucleotides have been shown in lymphocytes (Reichard, 1978,Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem. 57:349-374) toexert negative feedback on RR for pyrimidine dNTP synthesis but positivefeedback for purine dNTP synthesis, ensuring that the de novo synthesispathway is always active for the purines but is variable for thepyrimidines.

More importantly, the methods of the invention use stable isotope labelsinstead of radio-isotopes, and thus are safe for human use. Therefore, awide variety of uses are encompassed by the invention, including, butnot limited to, measurement of cellular proliferation and/or destructionin conditions where such information is of diagnostic value such ascancer, AIDS, hematologic disorders, endocrine disorders, bone disordersand organ failure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Biochemistry of DNA synthesis and routes of label entry. Not allintermediates are shown. G6P, glucose-6-phosphate; R5P,ribose-5-phosphate; PRPP, phosphoribosepyrophosphate; DNNS, de novonucleotide synthesis pathway; NDP, ribonucleoside-diphosphates; RR,ribonucleoside reductase; dN, deoxyribonucleoside; dNTP,deoxyribonucleoside-triphosphate; ³H-dT, tritiated thymidine; BrdU,bromodeoxyuridine.

FIG. 2. Overview of a specifically exemplified method for measurement ofDNA synthesis by incorporation of [6,6-²H₂] glucose.

FIG. 3. GC-MS of DNA digest (total ion current). Mass spectra of dA anddG peaks are shown as insets.

FIGS. 4A-4D. Labeling of tissue culture cells during log-phase growth invitro. Enrichment of dA from cellular DNA in hepatocyte (HepG2) (4A) andlymphocyte (H9) (4B) cell lines grown in media enriched with [6,6-²H₂]glucose. Lymphocyte data include results from experiments at twodifferent glucose enrichments. FIGS. 4C and 4D is a comparison offractional synthesis of DNA by HepG2 cells (4C) and H9 cells (4D),calculated from M₂ enrichments of dA/medium glucose, with increase incell numbers by counting.

FIGS. 5A-5B. Enrichment of dA from DNA of (5A) hepatocyte (HepG2) and(5B) lymphocyte (H9) cell lines grown in media containing 100% [6,6-²H₂]glucose for prolonged periods with repeated subcultures.

FIGS. 6A-6B. FIG. 6A demonstrates the enrichment of M₅ ion ofdeoxyadenosine from DNA of hepatocyte cell line (HepG2) cells grown inapproximately 20% [U-¹³C₆] glucose. FIG. 6B is a comparison offractional synthesis of DNA from M₅ labelling to proportion of new cellsby direct counting.

FIG. 7. Fractional synthesis of granulocytes in peripheral blood from 4subjects following two-day infusion of [6,6-²H₂] glucose, commencing attime zero. Open symbols, control subject; closed symbols, HIV-infectedsubjects. Fraction of new cells was calculated by comparison of dAenrichment to average plasma glucose enrichment, after correcting forestimated 35% intracellular dilution.

FIG. 8. Fractional synthesis of CD4⁺ lymphocytes obtained fromperipheral blood of an HIV infected patient following two-day infusionof [6,6-²H₂] glucose.

DETAILED DESCRIPTION OF THE INVENTION

The biochemical correlate of new cell production is DNA synthesis. DNAsynthesis is also relatively specific for cell division because“unscheduled” DNA synthesis is quantitatively minor (Sawada et al.,1995, Mutat. Res. 344:708-716). Therefore, measurement of new DNAsynthesis is essentially synonymous with measurement of cellproliferation.

The methods for measuring cell proliferation described herein haveseveral advantages over previously available methods. ³H-Thymidine is apotent anti-metabolite that has been used to kill dividing cells (Asheret al., 1995, Leukemia and Lymphoma 19:107-119); the toxicity ofintroducing radio-isotopes into DNA is avoided by the methods of theinvention which utilize stable isotopes. The toxicities of nucleosideanalogues, e.g. BrdU, are also avoided by labeling with a physiologicsubstrate through endogenous synthetic pathways.

Isotopic contamination by non-5-phase DNA synthesis is also minimized bylabeling through the de novo nucleotide synthesis pathway, which isprimarily active during S-phase. The variability of labeled pyrimidinenucleoside salvage uptake is resolved by labeling purine dNTP's via thede novo nucleotide synthesis pathway; turning what was previously adisadvantage (low purine dNTP labeling from the nucleoside salvagepathway) into an advantage (high and constant labeling from the de novopathway). This is demonstrated by the constancy of [6,6-²H₂] glucoseincorporation into DNA even in the presence of supraphysiologicextracellular concentrations of deoxyribonucleosides (Table 1). Possibleinput from free purine or pyrimidine base salvage does not dilute theribose moiety of NTP's, because the salvage pathway for free bases, likede novo synthesis of bases, involves combination with PRPP, which issynthesized from glucose (FIG. 1).

Moreover, re-utilization of label from catabolized DNA is avoided byanalyzing purine deoxyribonucleotides, because the deoxyribonucleosidesalvage pathway is low for purines. Die-away curves of labeled dA or dGin DNA after cessation of labeling will therefore be relativelyuncontaminated by isotope re-utilization, and cell turnover should bemeasurable from decay curves as well as incorporation curves(Hellerstein and Neese, 1992, Am J. Physiol. 263:E988-E1001).

Finally, the methods of the invention provide a quantitative measure forenumerating numbers of new cells as opposed to conventional methodswhich only detect the relative increase or decrease of cell numbers ascompared to controls.

Although the specific procedures and methods described herein areexemplified using labeled glucose as the precursor and detection of thelabel by analyzing purine deoxyribonucleosides, they are illustrativefor the practice of the invention. Analogous procedures and techniques,as well as functionally equivalent labels, as will be apparent to thoseof skill in the art based on the detailed disclosure provided herein,are also encompassed by the invention.

5.1. Stable Isotope Labels for Use in Labeling DNA During De NovoBiosynthesis of Nucleotides

The present invention relates to methods of measuring cellularproliferation by contacting a cell with a stable isotope label for itsincorporation into DNA via the de novo nucleotide synthesis pathway.Detection of the incorporated label is used as a measure of DNAsynthesis. Labeling DNA through the de novo nucleotide synthesis(endogenous) pathway has several advantages over conventional labelingmethods through the nucleoside salvage (exogenous) pathway. Theseinclude non-toxicity, specificity for S-phase of the cell cycle andabsence of re-incorporation of the label from catabolized DNA. The useof a non-radioactive label further reduces the risks of mutation.

In a specific embodiment illustrated by way of example in Section 6,infra, [6,6-²H₂] glucose, [U-¹³C₆] glucose and [2-¹³C₁] glycerol wereused to label the deoxyribose ring of DNA. Labeling of the deoxyriboseis superior to labeling of the information-carrying nitrogen bases inDNA because it avoids variable dilution sources. The stable isotopelabels are readily detectable by mass spectrometric techniques.

In a preferred embodiment of the invention, a stable isotope label isused to label the deoxyribose ring of DNA from glucose, precursors ofglucose-6-phosphate or precursors of ribose-5-phosphate. In embodimentswhere glucose is used as the starting material, suitable labels include,but are not limited to, deuterium-labeled glucose such as [6,6-²H₂]glucose, [1-²H₁] glucose, [3-²H₁] glucose, [²H₇] glucose, and the like;¹³C-1 labeled glucose such as [1-¹³C₁] glucose, [U-¹³C₆] glucose and thelike; and ¹⁸O-labeled glucose such as [1-¹⁸O₂] glucose and the like.

In embodiments where a glucose-6-phosphate precursor or aribose-5-phosphate precursor is desired, a gluconeogenic precursor or ametabolite capable of being converted to glucose-6-phosphate orribose-5-phosphate may be used. Gluconeogenic precursors include, butare not limited to, ¹³C-labeled glycerol such as [2-¹³C₁] glycerol andthe like, a ¹³C-labeled amino acid, deuterated water (2H₂O) and¹³C-labeled lactate, alanine, pyruvate, propionate or other non-aminoacid precursors for gluconeogenesis. Metabolites which are converted toglucose-6-phosphate or ribose-5-phosphate include, but are not limitedto, labeled (²H or ¹³C) hexoses such as [1-²H₁] galactose, [U-¹³C]fructose and the like; labeled (²H or ¹³C) pentoses such as [1-¹³C₁]ribose, [1-²H₁] xylitol and the like, labeled (²H or ¹³C) pentosephosphate pathway metabolites such as [1-²H₁] seduheptalose and thelike, and labeled (²H or ¹³C) amino sugars such as [U-¹³C] glucosamine,[1-²H₁] N-acetyl-glucosamine and the like.

The present invention also encompasses stable isotope labels which labelpurine and pyrimidine bases of DNA through the de novo nucleotidesynthesis pathway. Various building blocks for endogenous purinesynthesis may be used to label purines and they include, but are notlimited to, ¹⁵N-labeled amino acids such as [¹⁵N] glycine, [¹⁵N]glutamine, [¹⁵N] aspartate and the like, ¹³C-labeled precursors such as[1-¹³C₁] glycone, [3-¹³C₁] lactate, [¹³C]HCO₃, [¹³C] methionine and thelike, and ²H-labeled precursors such as ²H₂O. Various building blocksfor endogenous pyrimidine synthesis may be used to label pyrimidines andthey include, but are not limited to, ¹⁵N-labeled amino acids such as[¹⁵N] glutamine and the like, ¹³C-labeled precursors such as [¹³C]HCO₃,[U-¹³C₄] aspartate and the like, and ²H-labeled precursors (²H₂O).

It is understood by those skilled in the art that in addition to thelist above, other stable isotope labels which are substrates orprecursors for any pathways which result in endogenous labeling of DNAare also encompassed within the scope of the invention. The labelssuitable for use in the present invention are generally commerciallyavailable or can be synthesized by methods well known in the art.

5.2. Detection of Incorporated Label in DNA

The level of incorporation of stable isotope label into the DNA of cellsis determined by isolating the DNA from a cell population of interestand analyzing for isotope content a chemical portion of the DNA moleculethat is able to incorporate label from an endogenous labeling pathwayusing standard analytical techniques, such as, for example, massspectroscopy, nuclear magnetic resonance, and the like. Methods ofsample preparation will depend on the particular analytical techniquesused to detect the presence of the isotopic label, and will be apparentto those of skill in the art.

In a preferred embodiment of the invention, the presence of the label isdetected by mass spectrometry. For this method of detection, tissue orcells of interest are collected (e.g., via tissue biopsy, blood draw,collection of secretia or excretion from the body, etc.) and the DNAextracted using standard techniques as are well-known in the art. Ofcourse, the actual method of DNA isolation will depend on the particularcell type, and will be readily apparent to those of skill in the art.The cells may be optionally further purified prior to extracting the DNAusing standard techniques, such as, for example, immuno-affinitychromatography, fluorescence-activated cell sorting, elutration,magnetic bead separation, density gradient centrifugation, etc.

The DNA is then hydrolyzed to deoxyribonucleosides using standardmethods of hydrolysis as are well-known in the art. For example, the DNAcan be hydrolyzed enzymatically, such as for example with nucleases orphosphatases, or non-enzymatically with acids, bases or other methods ofchemical hydrolysis.

Deoxyribonucleosides are then prepared for mass spectrometric analysisusing standard techniques (e.g., synthesis of trimethylsilyl, methyl,acetyl, etc. derivatives; direct injection for liquid chromatography;direct probe sample introduction, etc.) and the level of incorporationof label into the deoxyribonucleosides determined.

The mass spectrometric analysis is of fragment potentially containingstable isotope label introduced from endogenous labeling pathway. Forexample, the m/z 467-469 fragment of the deoxyadenosine or the m/z 557and 559 fragment of the deoxyguanosine mass spectrum, which contain theintact deoxyribose ring, could be analyzed after [6,6-²H₂] glucoseadministration, using a gas chromatograph/mass spectrometer underelectron impact ionization and selected ion recording mode. Or the m/z103 and 104 fragment of the deoxyadenosine mass spectrum, which containsthe position C-5 of deoxyribose, could be analyzed after administrationof [6,6-²H] glucose or [6-¹³C₁] glucose. In a preferred embodiment, themass spectrometric fragment analyzed is from purine deoxyribonucleosidesrather than pyrimidine deoxyribonucleosides.

The fraction of newly synthesized DNA and therefore newly divided cells(cell proliferation or input rate) or newly removed cells (cell death orexit rate) is then calculated (Table 1). TABLE 1 abundances abundancesm/z m/z dA* f (uncorrected) f (corrected) Day # 457 459 enrichment (%new cells) (% new cells) 1 2844049 518152 0.00000 0.00 0.00 (Baseline) 21504711 260907 0.00000 0.00 0.00 3 2479618 453609 0.00298 2.50 3.84 43292974 624718 0.00586 4.91 7.55 5 2503144 461905 0.00451 3.77 5.81 61055618 186087 0.00318 2.66 4.09 7 2186009 394058 0.00193 1.61 2.48 82154805 389390 0.00250 2.09 3.22 9 2472480 455139 0.00417 3.49 5.38 101532187 272195 0.00238 2.00 3.07Abundances represent average of three acquisitions.Plasma glucose enrichment=11.9%; dA*=deoxyadenosine enrichment based oncomparison to abundance corrected standard curve of[5,5-²H₂]deoxyadenosine; f uncorrected, calculated as dA enrichmentdivided by plasma glucose enrichment; f corrected, calculated as dAenrichment divided by 0.65 times plasma glucose enrichment

5.3. Uses

5.3.1. In Vitro Uses

In a specific embodiment illustrated by way of example in Section 6,infra, an enrichment of deoxyadenosine (dA) was observed in two celltypes incubated with a stable isotope label and grown as monolayers andin suspension. The dA enrichment correlated closely with the increase incell numbers by direct counting. Therefore, the methods of the inventionmay be used to measure cellular proliferation in a variety ofproliferative assays. For instance, bioassays which use cellularproliferation as a read-out in response to a growth factor, hormone,cytokine or inhibitory factor may be developed by using a stable isotopelabel which targets the de novo nucleotide synthesis pathway. Examplesof such assays include lymphocyte activation by antigen andantigen-presenting cells, apoptosis of target cells induced by tumornecrosis factor and cytotoxicity of tumor cells by cytolyticlymphocytes.

5.3.2. In Vivo Uses

Since the methods of the invention do not involve radioactivity andpotentially toxic metabolites, these methods are particularly useful asa diagnostic tool in measuring cellular proliferation and destructionrates in vivo. In comparison to conventional methods in humans, themethods of the invention are safe, more widely applicable, more easilyperformed, more sensitive and produce more accurate results because thede novo nucleotide synthesis pathway is constant and predominant, is notdiluted, label via physiologic substrates rather than potentially toxic,non-physiologic metabolites, do not require preservation of cell ortissue anatomy and involve no radioactivity.

A wide variety of medical applications in which cellular proliferationand destruction play an important role are encompassed by the presentinvention. In particular the methods of the invention may be used todetermine the proliferation and destruction rates in cancer, infectiousdiseases, immune and hematologic conditions, organ failure and disordersof bone, muscle and endocrine organs.

5.3.2.1. Cancer Treatments

In one embodiment of the invention, a patient could receive systemic orlocal administration of a stable isotope labeled precursor for the denovo nucleotide synthesis pathway (e.g., [6,6-²H₂] glucose at 1.25 g/hrfor 24-48 hr intravenously) prior to initiation of chemotherapy, andagain 1-2 weeks after starting chemotherapy. A small specimen of theturnover (e.g., by skinny needle aspiration) is performed after eachperiod of stable isotope administration in the synthesis rate of DNA,reflecting inhibition of tumor cell division, could be used as atreatment end-point for selecting the optimal therapy. Typically, thedose of isotope precursor given is enough to allow incorporation intodeoxyribonucleosides above the mass spectrometric detection limits.Samples are taken depending on tracer dilution and cell turnover rates.

5.3.2.2. Cancer Prevention

The risk for breast, colon and other cancers tends to vary withproliferative stress in the tissue, i.e., hormones, inflammation ordietary factors that alter cell proliferation profoundly affect cancerrates. The ability to characterize a woman's underlying mammary cellproliferative stress and its response to preventative intervention inearly adult life, for example, would radically alter breast cancerprevention. The same applies to colon cancer, lung cancer, and othercancers.

5.3.2.3. AIDS

Anti-retrovirals in AIDS are intended to block viral replication (abiosynthetic process) in order to reduce CD4+ T cell death and turnover.Recent advances in AIDS treatment have focused precisely on thesekinetic processes, although direct kinetic measurements were notavailable. The ability to measure directly these treatment end-pointscan radically change the nature of HIV therapeutics. Physicians canquickly determine whether to begin aggressive anti-retroviral treatmentearly in the disease for each individual patient. In a specificembodiment illustrated by way of example in Section 6, infra, themethods of the invention are used to accurately measure theproliferation and destruction rates of CD4⁺ cells in HIV patients.

5.3.2.4. Conditions in which Cellular Proliferation is Involved

A large number of conditions are known to be characterized by alteredcellular proliferation rates and thus can be monitored by methods of theinvention:

Cancer: Malignant tumors of any type (e.g., breast, lung, colon, skin,lymphoma, leukemia, etc.); pre-cancerous conditions (e.g., adenomas,polyps, prostatic hypertrophy, ulcerative colitis, etc.); factorsmodulating risk for common cancers (e.g., estrogens and breastepithelial cells; dietary fat and colonocytes; cigarette smoking oranti-oxidants and bronchial epithelial cells; hormones and prostatecells, etc.).

Immune disorders: CD4⁺ and CD8⁺ T lymphocytes in AIDS; T and Blymphocytes in vaccine-unresponsiveness; T cells in autoimmunedisorders; B cells in hypogammaglobulinemias; primary immunodeficiencies(thymocytes); stress-related immune deficiencies (lymphocytes); and thelike.

Hematologic conditions: White blood cell deficiencies (e.g.,granulocytopnia); anemias of any type; myeloproliferative disorders(e.g., polycythemia vera); tissue white cell infiltrative disorders(e.g., pulmonary interstitial eosinophilia, lymphocytic thyroiditis,etc.); lymphoproliferative disorders; monoclonal gammopathies; and thelike.

Organ failure: Alcoholic and viral hepatitis (liver cells); diabeticnephropathy (glomerular or mesangeal cells); myotrophic conditions(myocytes); premature gonadal failure (oocytes, stromal cells of ovary,spermatocytes, Leydig cells, etc.); and the like.

Conditions of bone and muscle: Response to exercise training or physicaltherapy (myocytes or mitochondria in myocytes); osteoporosis(osteoclast, osteoblasts, parathyroid cells) myositis; and the like.

Endocrine conditions: Diabetes (islet B-cells); hypothyroidism andhyperthyroidism (thyroid cells); hyperparathyroidism (parathyroidcells); polycystic ovaries (stromal cells of ovary); and the like.

Infectious diseases. Tuberculosis (monocytes/macrophages); bacterialinfections (granulocytes); abscesses and other localized tissueinfections (granulocytes); viral infections (lymphocytes); diabetes footdisease and gangrene (white cells); and the like.

Vascular disorders. Atherogenesis (smooth muscle proliferation inarterial wall); cardiomyopathies (cardiac myocyte proliferation); andthe like.

Occupational diseases and exposures. Susceptibility to coal dust forblack lung (fibroblast proliferative response); susceptibility to skindisorders related to sun or chemical exposures (skin cells); and thelike.

The isotope label suitable for use in vivo is prepared in accordancewith conventional methods in the art using a physiologically andclinically acceptable solution. Proper solution is dependent upon theroute of administration chosen. Suitable routes of administration may,for example, include oral, rectal, transmucosal, transcutaneous, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

Alternatively, one may administer a label in a local rather thansystemic manner, for example, via injection of the label directly into aspecific tissue, often in a depot or sustained release formulation.

Determination of a detectable amount of the label is well within thecapabilities of those skilled in the art.

EXAMPLE Measurement of Cell Proliferation by Labeling DNA with StableIsotope-Labeled Glucose

6.1. Materials and Methods

6.1.1. Isolation of Deoxyribonucleosides from DNA

DNA was prepared from cells or tissues by phenol-chloroform-isoamylalcohol extraction of cell suspensions or tissue homogenates. Yield andpurity were confirmed by optical density. After heat denaturation, DNAwas hydrolyzed enzymatically to deoxyribonucleosides by sequentialdigestion with nuclease P1, phosphodiesterase, and alkaline phosphatase,as described by Crain et al. (Crain, 1990, Methods Enzymol.193:782-790). Nucleoside yield and purity ere confirmed by HPLC using aC-18 column and water-methanol gradient (Shigenaga et al., 1994, MethodsEnzymol. 234:16-33).

6.1.2. Derivatization of Deoxyribonucleosides and Analysis by GasChromatography Mass Spectrometry (GC-MS)

Trimethylsilyl derivatives of nucleosides were synthesized by incubationof lyophilized hydrolysates with BSTFA: pyridine (4:1) at 100° C. for 1hour. Samples were analyzed by GC-MS (DB-17 HT column, J&W Scientific,Folsom Calif.; HP 5890 GC and 5971MS, Hewlett Packard, Palo Alto,Calif.). Abundances of ions at mass to charge ratio (m/z) 467 and 469were quantified under selected ion recording mode for deoxyadenosine(dA); and m/z 555 and 557 were monitored for deoxyguanosine (dG). Underthe derivatization and GC-MS conditions employed, the purines (dA anddG) gave larger peaks (FIG. 3) than the pyrimidines, resulting ingreater sensitivity and a higher signal to noise ratio. To measure theenrichment of glucose, plasma or culture media were deproteinized withperchloric acid and passed through anion and cation exchange columns(Neese et al., 1995, J. Biol. Chem. 270:14452-14463). The glucosepenta-acetate derivative was formed by incubation with acetic anhydridein pyridine. GC-MS analysis was performed as described previously (Neeseet al., 1995, J. Biol. Chem 270:14552-14663), monitoring m/z 331 and 333under selected ion recording.

6.1.3. In Vitro Studies

Initial studies of label incorporation from [6,6-²H₂] glucose intocellular DNA were performed in tissue culture cell lines. Two cell lineswere used: a hepatocyte cell line, HepG2, and a lymphocytic cell line,H9, which is a CD4⁺ T cell line. HepG2 cells were grown in 10 ml disheswith alpha-modified Dulbecco's minimum essential media (MEM). H9 cellswere grown in suspension in RPMI 1640. Both were grown in the presenceof 10% dialyzed fetal calf serum and antibiotics (all reagents wereobtained from Gibco-BRL, Gaithersburg, Md., except where stated). Inboth cases the number of cells present was measured by counting analiquot on a Coulter ZMO901 cell counter. For HepG2 cells, platingefficiency was corrected for by counting an identical plate at thebeginning of each labeling phase. Cells were labeled by addition of[6,6-²H₂] glucose (Cambridge Isotope Laboratories, Andover, Mass.) suchthat labeled glucose constituted 10-20% by weight of total glucosepresent (100 mg/L for MEM-a and 200 mg/L for RPMI 1640). In someexperiments, glucose free medium was used and only 100% labeled glucosewas present in the medium. Additional experiments were carried out inthe presence of [U-¹³C₆] glucose and [2-¹³C₁] glycerol (CambridgeIsotope Laboratories, Andover, Mass.).

6.1.4. Animal Studies

Four rats (approximately 250 g) were infused with labeled glucose.Intravenous canulae were placed under anesthesia (Hellerstein et al.,1986, Proc. Natl. Acad. Sci. USA 83:7044-7048). After a 24-48 hrrecovery period, [6,6-²H₂] glucose was infused as a sterile 46 mg/mlsolution at 0.5 ml/hr for approximately 24 hours. Food was withdrawn atthe beginning of the isotope infusion. This dose was expected to achieveaverage plasma glucose enrichments of about 10%, based on previousstudies in fasting rats (Neese et al., 1995, J. Biol. Chem.270:14452-14463). At the end of the infusion period, animals weresacrificed. Blood for plasma glucose enrichment and tissues for DNAextraction were collected and frozen prior to analysis. A section of theintestine approximately 30 cm in length was excised from just below theduodenum in each rat. The intestinal segments were everted and washed.Epithelial cells were released from the submucosa by incubation withshaking in buffer containing 5 mM EDTA at 37° C. for 10 min, asdescribed by Traber et al. (Traber et al., 1991, Am. J. Physiol.260:G895-G903). DNA was extracted from cell preparations, thenhydrolyzed to nucleosides and analyzed by GC-MS (FIG. 2).

6.1.5. Studies of Granulocyte Kinetics in Human Subjects

In order to investigate the application of the methods of the inventionin clinical settings, four volunteers received intravenous infusion of[6,6-²H₂] glucose (60 g over 48 h) in the General Clinical ResearchCenter at San Francisco General Hospital. One subject was a healthynormal volunteer and the other three were HIV-seropositive men, who wereparticipating in lymphocyte kinetic studies (blood CD4 cell counts inthe range 215-377/mm³). None had a clinically apparent infection at thetime of the infusion. In order to enable high and relatively constantenrichments of plasma glucose and maximize labeling of cellular DNA,dietary carbohydrate was restricted (mean intake 46 g/d) during the2-day period of the infusion. A heparinized blood sample was drawn atbaseline and every 12 hr during the infusion, for estimation of plasmaglucose enrichment. After the 48-hr infusion, blood was collected dailyfor 10 days and granulocytes and mononuclear cells were separated bygradient centrifugation (Vacutainer CPT, Becton Dickinson, FranklinLakes, N.J.). Granulocyte DNA was extracted, hydrolyzed to nucleosidesand analyzed by GC-MS, as described in Section 6.1.2, supra. Allprocedures received prior approval by the University of California atSan Francisco Committee on Human Research and the University ofCalifornia at Berkeley Committee for the Protection of Human Subjects,and written informed consent was obtained from subject for allprocedures carried out.

6.2. Results

6.2.1. Development of Analytic Method

Derivatization is required to volatilize deoxyribonucleosides for GC-MSanalysis (Blau and Halket, 1993, Handbook of Derivatives forChromatography 2nd ed.). The highest abundances with TMS derivatizationwas observed, compared to methylation or acetylation. The GC-MS scans ofa typical TMS-derivatized sample, analyzed under electron impactionization, are shown (FIG. 3). dA and dG eluted from the GC columnshowed well defined peaks. As described previously (McCloskey, 1990,Methods Enzymol. 193:825-841), the dominant ions in the spectra werefrom the base moiety that were unlabeled from [6,6-²H₂] glucose. Theparent ions, m/z 467 and 557 for dA-TMS₃ and dG-TMS₄, respectively, werewell represented and were present in a region of the mass spectrum withlittle background. Labeled samples contained an excess of the M+2 ions469 and 557; the ratios of 469 to 467 and 557 to 559 were used forquantification.

Abundance sensitivity of isotope ratios (concentration dependance) wasobserved for dA and dG, as described for GC-MS (Neese et al., 1995, J.Biol. Chem. 270:14452-14463; Patterson and Wolfe, 1993, Biol. MassSpectrom 22:481-486). Samples were therefore always analyzed atabundances matched to those in the standards used for baseline (naturalabundance) subtraction, when calculating isotope enrichments. Inenriched samples, the measured enrichments of dA were not significantlydifferent from dG, as expected. Only data from dA are shown below.

6.2.2. In Vitro Cell Proliferation Studies

The enrichment of dA derived from cells grown in media containing 10-15%[6,6-²H₂] glucose increased progressively with time (FIGS. 4A and 4B).This was demonstrated for both a hepatocyte cell line (HepG2) grown asmonolayers on plates, and for a T-lymphocytic cell line (H9) grown insuspension. When compared to the number of cells measured by directcounting, dA enrichment correlated closely with the increase in cells bydirect counting (FIGS. 4C and D). The correlation coefficient betweenthe fraction of new DNA (calculated from the ratio of M2 enrichments indA to medium glucose) and the percentage of new cells by direct countingwas 0.984 with HepG2 cells and 0.972 for H9 cells.

The enrichment of the true intracellular dATP precursor pool for DNAsynthesis using growing cells was equal in theory to the dA enrichmentin DNA at 100% new cells (i.e., when only labeled DNA was present).Extrapolation of the labeling time course experiments to 100% new cellsgave estimated plateau dA enrichments of 0.725 of the medium glucoseenrichment for HepG2 cells and 0.525 for H9 cells (FIGS. 4A-D).

In order to test directly the relationship between enrichments ofextracellular glucose and intracellular DNA precursors, cells were grownfor prolonged periods in medium containing 100% [6,6-²H₂] glucose withrepeated replating or subculture of cells, for a total of 53 days forHepG2 cells and 25 days for H9 cells. At the end of the experiment,<0.1% of DNA present could be accounted for by the initial unlabeledcells. Maximum enrichment of dA was about 65% for both HepG2 and H9cells (FIGS. 5A and B). One possible explanation for this dilution ofextracellular labeled glucose could be the synthesis of glucose withinthe cell, e.g. from gluconeogenesis (GNG), since unlabeled amino acidprecursors for GNG were present in the culture medium. Alternatively,some exchange of the label might have occurred during intracellularmetabolism of glucose, either during glycolysis and passage through thetricarboxylic acid cycle or during the non-oxidative portion of thepentose-phosphate pathway (Wood et al., 1963, Biochemische Zeitschrift338:809-847).

If intracellular unlabeled glucose from GNG were the dominant origin ofdilution, dA from H9 cells might approach closer than the Hep G2 cellsto 100% of medium glucose enrichment. However, this was not found to bethe case (FIGS. 4A-D and 5A and B). A more direct test would be theincorporation of GNG precursors into dA in DNA by HepG2 cells. In orderto test this hypothesis, both HepG2 and H9 were cultured in the presenceof [2-¹³C₁] glycerol. By applying the theory of combinatorialprobabilities, or the mass isotopomer distribution analysis (MIDA)technique (Neese et al., 1995, J. Biol. Chem. 270:14452-14463;Hellerstein et al., 1992, Am. J. Physiol. 263:E988-E1001), the fractionof deoxyribose in dA that came from GNG could then be calculated. WhenHepG2 cells were grown in media to which [2-¹³C₁] glycerol had beenadded at concentration of 20 μg/ml, negligible incorporation of ¹³C intodA was found. In the presence of 100 μg/ml [2-¹³C₁] glycerol(approximately 2-3 times plasma glycerol concentrations), enrichment ofboth M₊₁ and M₊₂ ions was observed in dA. Applying MIDA revealed that17.8% of dA pentose ring synthesis appeared to arise from GNG ratherthan utilization of extracellular glucose. H9 cells grown in thepresence of 100 μg/ml [2-¹³C₁] glycerol revealed no measurable GMG, asexpected.

Duplicate pairs of cell cultures were also grown in the presence of 10%[6,6-²H₂] glucose with and without the addition of unlabeled glycerol(100 μg/ml). Such unlabeled glycerol did not affect labeling in H9cells; in HepG2 cells, incorporation into dA was reduced by 7%. Thus, itappears that the availability of GNG precursors had only a small effecton the labeling of DNA in cells capable of GNG and GNG does not fullyexplain the intracellular dilution of dA.

If the roughly 35% dilution between extracellular glucose and dA in DNAwere due to exchange of ²H for ¹H in intracellular glucose cycles,carbon label in [U-¹³C₆] glucose should undergo re-arrangement.Accordingly, HepG2 and H9 cells were grown in the presence of 10%[U-¹³C₆] glucose. If there were no metabolism through pathways such asthe non-oxidative portion of the pentose phosphate pathway, the dNTP'sfrom this precursor should retain all five labeled carbons and have amass of M₅. The M₅ enrichment increased in a similar fashion to thatobserved with the M₂ ion from [6,6-²H₂] glucose. An asymptote ofapproximately 80% of the extracellular enrichment was reached in HepG2cells (FIGS. 6A and B) while in H9 cells the asymptote was approximately60% of extracellular glucose enrichment. When the M₀ to M₅ spectrum wasanalyzed, enrichments of M₂, M₃, and M₄ ions were seen in addition tothe expected enrichment of M₅. This phenomenon was observed in both H9and HepG2 cells, although the relative abundance of these ions wasgreater from H9 cells.

The above cell culture experiments were performed in the absence ofdeoxyribonucleosides in the medium. Previous studies with lymphocytecell lines (Reichard, 1978, Fed. Proc. 37:9-14; Reichard, 1988, Ann.Rev. Biochem. 57:349-374) have suggested that increasing theavailability of extracellular deoxyribonucleosides does not reduce, andmay even increase, activity of ribonucleoside reductase and theendogenous synthesis pathway for purine dNTP's (FIG. 1). To testdirectly the effects of increased availability of extracellulardeoxyribonucleosides, HepG2 and H9 cells were grown in the presence ofan equimolar mixture of the four deoxyribonucleosides. Twoconcentrations, 20 and 100 μM, were chosen to reproduce or exceed thoseprevailing in tissues; plasma concentrations are normally of the orderof 1 μM and tissue concentrations may range between 1 and 100 μM (Cohenet al., 1983, J. Biol. Chem. 258:12334-12340). Six flasks of H9 cellswere grown in parallel in media labeled with ca. 10% [6,6-²H₂] glucose(Table 2). TABLE 2 Effect of extracellular deoxyribonucleosides onincorporation of [6,6-²H₂] glucose into dA in DNA ExtracellularDeoxyribonucleoside Concentration (μM) 0 0 20 20 100 100 Lymphocytes(H9) dA/glucose ratio 0.527 0.522 0.535 0.528 0.534 0.514 Fraction newcells 0.849 0.851 0.867 0.856 0.839 0.822 (by counting) Extrapolated0.620 0.613 0.617 0.617 0.637 0.626 dA/glucose (100% new cells)Hepatocytes (HepG2) dA/glucose ratio 0.386 0.385 0.381 0.370 0.339 0.344Fraction new cells 0.568 0.589 0.536 0.565 0.626 0.570 (by counting)Extrapolated 0.680 0.653 0.711 0.655 0.541 0.603 dA/glucose (100% newcells)Two were grown without added deoxyribonucleosides, two were grown at thelower and two at the higher deoxyribonucleoside concentrations. After 90hours, 85% of cells were new by counting. The experiment was alsoperformed with HepG2 cells, yielding an average increase in cell numberrepresenting about 58% new cells. In H9 cells the presence ofextracellular deoxyribonucleosides at either 20 or 100 μM did not reducethe incorporation of label from glucose into dA and thus did not appearto suppress the activity of the de novo nucleotide synthesis pathway. InHepG2 cells there was no appreciable reduction in incorporation at 20μM, although there was a small (ca. 12%) reduction at 100 μM. In H9cells, the extrapolated ratio of dA/glucose at 100% new cells (based oncell counting) was reproducibly between 62-64%. For HepG2 cells, theratio ranged between 54-71%.

6.2.3. In Vivo Labeling of DNA: Animal Studies

In rats (n=4) receiving intravenous infusion of [6,6-²H₂] glucose,plasma glucose enrichment at sacrifice was 13.2±0.9%. The mean plasmaglucose enrichment for the whole 24 hour infusion period was less thanthis value because plasma glucose enrichment progressively increasedduring fasting, as the Ra glucose progressively fell (Rocha et al.,1990, Eur. J. Immunol. 20:1697-1708). The mean plasma glucose enrichmentwas estimated from two rats receiving labeled glucose infusion, and inwhich repeated blood samples were taken via arterial blood-drawing line.The mean enrichment for the 24 h fasting period was 0.70 of the finalenrichment; accordingly, this value was used for calculating the meanglucose enrichments for the four experimental rats (9.2±0.6%).

Differing enrichments were found in dA from the three tissues studied(Table 3). TABLE 3 In vivo incorporation of [6,6-²H₂] glucose into dA inDNA in various tissues in rats dA Enrichment Percent New Cells Turnovertime (d) Rate Constant, K (d⁻¹) t_(1/2)(d) Tissue (%) UncorrectedCorrected Uncorrected Corrected Uncorrected Corrected UncorrectedCorrected Intestinal 3.18 ± 0.24 34.6 ± 4.2 53.2 ± 6.5 2.83 ± 0.37 1.84± 0.24 — — — — epithelium Thymus 2.33 ± 0.08 25.3 ± 2.2 38.9 ± 3.1 — —0.302 ± 0.030  0.51 ± 0.058 2.31 ± 0.23 1.36 ± 0.15 Liver 0.25 ± 0.06 2.7 ± 0.5  4.2 ± 0.9 — — 0.028 ± 0.005 0.044 ± 0.008 25.4 ± 4.9  16.2 ±3.1 Mean plasma glucose enrichment was 9.2 ± 0.6% and mean duration ofinfusion was 23.2 ± 0.1 hr. Uncorrected calculations use plasma glucoseenrichments as precursor for DNA synthesis; corrected calculations use acorrection factor of 0.65 × plasma glucose enrichment to account fordilution in the intracellular precursor pool (see text). A linearkinetic model was used for intestinal epithelium; an exponential modelwas used for thymus and liver.For intestinal epithelial cells, a life-span (linear) kinetic model wasemployed based on the assumption that new cells divided, lived for afixed period of time and then died in the order in which they wereformed, representing the progression of intestinal epithelial cells fromcrypt to villus tip (Lipkin, 1987, Physiol. Gastrointest. Tract, pp.255-284, Ed. Johnson, L. R.). A turnover time of 2.8±0.4d was calculated(uncorrected, using plasma glucose enrichments to representintracellular DATP) or 1.8±0.2d (corrected, using plasma glucose with a35% intracellular dilution factor). For thymus and liver a randomreplacement (exponential) model was applied. Thymus had a 10-times morerapid turnover than liver (Table 3).

6.2.4. Clinical Studies of Granulocyte Kinetics in Human Subjects

As part of a study of T lymphocyte kinetics in AIDS, three HIVseropositive men and one HIV seronegative man received an infusion of[6,6-²H₂] glucose (1.25 g/hr for 48 hr). All were clinically stable atthe time of investigation. Absolute granulocyte counts were 1.5, 0.9,and 2.4×10⁹/L, respectively, in the HIV-positive subjects and 2.4×10⁹/Lin the control subject. The infusions were well tolerated. Mean plasmaglucose enrichments of 15.3±2.4 molar percent excess were achieved (rateof appearance of glucose about 2 mg/kg/min). Granulocytes were isolatedand dA enrichment measured from DNA. For the first 6 days following theinfusion, very low proportions of labeled cells were seen in thecirculation (FIG. 7), followed by the appearance of labeled cellsstarting on days 6-8. Enrichments at day 8 indicated about 25% new cellspresent (corrected).

6.2.5. Measurement of T Cell Proliferation in HIV Infection

T cell proliferation rates in individuals infected with humanimmunodeficiency virus (HIV) was measured by the methods of theinvention. An intravenous infusion of [6,6-²H₂] glucose was performed inmen with well-maintained CD4⁺ T cell numbers (>500/mm³) or low CD4+counts (<200/mm³). The infusion was for 48 hr at 1.25 g [6,6-²H₂]glucose/hr, to achieve 10-15% proportion of labeled glucose molecules inthe blood plasma (10-15% enrichment). Blood (20-30 cc) was collecteddaily during the infusion and for the following 10 days.

Mononuclear cells were isolated using PT® tubes; and CD4⁺ cells werethen isolated using either magnetic bead immunoseparation (Dynabeads®)or fluorescent activated cell sorting to isolate 10⁶ cells. DNA fromisolated cells was recovered using a commercial kit (Quiagen®). DNA washydrolyzed to free deoxyribonucleosides enzymatically with nuclease P₁,phosphodiesterase, and alkaline phosphatase; the hydrolysate wasderivatized with FSTFA to the trimethylsilyl derivatives ofdeoxyribonucleosides, which were injected into a table top GC-MSinstrument. The dA and dG peaks from the GC effluent were monitored byselected ion recording mass spectrometry and m/z 467 and 469 (for dA)and m/z 555 and 557 (for dG) were quantified, through comparison tostandard curves of commercially labeled material analyzed concurrently(e.g., [5,5-²H₂] dA purchased from CIL, Cambridge, Mass.). Standardcurves were abundance matched between standards and sendes to correctfor concentration sensitivity of isotope ratios. Enrichments in dA anddG from CD4⁺ and CD8⁺ T cells were in the rate 0.00 to 1.50 percentlabeled species. By comparison to the plasma glucose isotope enrichment(10-15 percent labeled species) with a 35% dilution correction andapplication of the precursor-product relationship (Hellerstein andNeese, American Journal of Physiology 263:E988-1001, 1992), thepreparation of newly synthesized DNA strands was quantified. Peak valueswere at 2-3 days after the start of [6,6-²H₂] glucose infusions andreached 15-20% newly synthesized DNA strands, and thus 15-20% newlyproliferating cells (FIG. 8). The die away curves of dA or dG labelingbetween days 4 and 10 revealed the destruction rate of the label andtherefore recently dividing population of cells (Hellerstein and Neese,1992, American Journal of Physiology 263: E988-1001). Destruction ratesof labeled cells was generally higher than for the general population ofcells implying selective death of recently divided and activated cells.The effect of CD4⁺ T cell proliferation and destruction ofanti-retroviral therapies was then determined, by repeating the[6,6-²H₂] glucose infusion after 8-12 weeks of therapy.

In conclusion, a method for measuring DNA synthesis using stable isotopelabels and mass spectrometry was developed for measuring cellproliferation. This method involves no radioactivity and potential toxicmetabolites, and is thus suitable for use in humans.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention and any sequences which are functionally equivalent are withinthe scope of the invention. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

All publications cited herein are incorporated by reference in theirentirety.

1-49. (canceled)
 50. A method for analyzing metabolic pathways,comprising: a. administering to a subject a substrate labeled with astable isotope, wherein the relative isotopic abundance of the isotopein the substrate is known; b. allowing the labeled substrate to be atleast partially metabolized by the subject to form one or more targetmetabolites; and c. determining the abundance of the isotope in aplurality of target analytes in a sample from the subject to determine avalue for the flux of each target analyte, wherein the plurality oftarget analytes comprise the substrate and/or one or more of the targetmetabolites.